The present invention is directed to a process for the separation and purification of DNA, and, more specifically, to processes for the separation of the chromosome fragments thereof. In one embodiment of the present invention, a process is provided for separating DNA fragments of any size. In another embodiment of the present invention, a process is provided which comprises providing a mixture of DNA fragments of desired sizes, depositing the fragments in a conventional gel electrophoresis apparatus, and applying a series of unidirectional field pulses across the gel, thereby enabling separation of the fragments according to their sizes. Another embodiment of the invention comprises the selection of a computer program that simulates a DNA gel electrophoresis process.
In the field of genetic engineering, DNA is typically studied by severing long DNA chains into smaller fragments using a restriction enzyme. The resulting fragments, which must then be separated according to size or composition, provide the information needed to construct a map of the original DNA chain. Construction of such a map is facilitated by severing the original DNA chain into a relatively small number of long fragments (preferably less than one hundred), as opposed to generating many short fragments. Also, as the number of pieces decreases, it becomes easier to reconstruct the original molecule. Conventional methods of fragment separation are limited in the mixtures containing fragments having more than 20,000 base pairs cannot readily or fully be separated. Therefore, with, for example the conventional methods of separation, it is required to cut human chromosomes into thousands of fragments to permit the separation thereof, thus reconstruction of the original chain would be extremely difficult.
In an attempt to alleviate some of these difficulties, variations on the standard known electrophoresis method have been developed. For example, there is described in U.S. Pat. No. 4,473,452, the disclosure of which is totally incorporated herein by reference, a pulsed field gradient gel electrophoresis method, which involves the application of two nonuniform electric fields positioned at approximately right angles to each other as a means of separating DNA fragments of over 20,000 base pairs. In addition, according to the abstract of the '452 patent, there is recited an apparatus for and a method of electrophoretically separating particles by electric fields which are approximately transverse to each other, and alternate between respectively high and low intensities out of phase with each other at a frequency related to the mass of the particles, thus permitting movement of the particles in an overall direction intermediate between the respective directions of the fields. In addition, this patent discloses the use of pulsed and crossed gradient electric fields to separate and resolve DNA fragments of up to several million base pairs. In contrast, with the process of the present invention, for example, several uniform fields are selected and applied in pulses in a single direction for the purpose of separating DNA fragments of any size.
Also, the '452 patent discloses that particularly good results are obtained when the on and off times of the alternate fields are proportional to the mass of the particles to be separated raised to a power of about 1.5. More specifically, this patent illustrates that the proper choice of a frequency at which the change from one field to another should occur is related to the time it takes the particle (molecule) of interest to orient itself into an elongated cylindrical shape, and that this time t is related to the mass of the particle (the molecular weight) M, the effective pore radius of the gel r, and the measured velocity of the particle in the gel v, in accordance with the relationship: EQU t.alpha.M.sup.1.5 /(r.sup.2 v)
Additionally, in the '452 patent it is indicated that variations on the invention, such as a differently shaped electrophoresis chamber, or differently produced, distributed or varied electric fields can be used provided that the particles are acted on by electric fields varying with time, permitting them to move in overall directions generally intermediate between at least two of the relevant, operationally significant fields. Moreover, more than two fields can be used providing the net effect is at least to act in the desired manner on a particle first in one direction, then in another direction transverse to the first, thereby moving the particle in a third direction intermediate between the first two. Thus, the process of the '452 patent requires the use of crossed alternating gradient fields to separate large DNA fragments.
The variation on standard electrophoresis process presented in the '452 patent is also discussed by C. L. Smith and C. R. Cantor in "Pulsed-Field Gel Electrophoresis of Large DNA Molecules," Nature, Vol. 319, pages 701-702 (1986), and by L. M. Corcoran in "Molecular Karyotypes: Separating Chromosomes on Gels," BioEssays, Vol. 3, No. 6, pages 269-271 (1985), the disclosure of each of these articles being totally incorporated herein by reference. Another modification of the standard electrophoresis method is disclosed by G. F. Carle, M. Frank, and M. V. Olson in "Electrophoretic Separations of Large DNA Molecules by Periodic Inversion of the Electric Field," Science Reports, Vol. 232, pages 65-68 (1986), the disclosure of which is totally incorporated herein by reference. This article discloses a method for the separation of DNA fragments containing 15,000 to over 700,000 base pairs by periodically inverting a uniform electric field of a given strength in one dimension.
In an article by R. G. Snell and R. J. Wilkins entitled "Separation of Cromosomal DNA Molecules from C. albicans by Pulsed Field Gel Electrophoresis," Nucleic Acids Research, Vol. 14, No. 11, pages 4401-4406 (1986), the disclosure of which is totally incorporated herein by reference, the authors discuss the method of separation disclosed in the '452 patent. The article indicates that variations in experimental conditions such as pulse time, temperature, and relative voltage conditions have critical effects on the quality of results, and that pulsed field gel electrophoresis can be used to resolve DNA from chromosomes of the Candida albicans and Saccharomyces cerrevisiae strains of yeast. According to the aforementioned article, the single most important factor for obtaining optimal resolution was the elevation of the electrophoresis temperature to 35.degree. C. Alteration of relative voltage conditions by 10 percent, pulse time by 20 percent, or temperature by 10 percent was, according to this article, found to destroy the electrophoretic pattern.
"Dependence of the Electrophoretic Mobility of DNA in Gels on Field Intermittency," T. Jamil and L. S. Lerman, Journal of Biomolecular Structure and Dynamics, Vol. 2, No. 5, pages 963-966 (1985), the disclosure of which is totally incorporated herein by reference, addresses the effect of varying pulse duration and varying the interval between pulses upon the mobility of DNA fragments in gels. This article illustrates the mobility of lambda DNA fragments containing from 3,400 to 21,800 base pairs when a single pulsed field is applied. The authors concluded that if the interval between pulses remains constant, the apparent mobility increases as the duration of pulses increases; however, it approaches a maximum. Additionally, this article discloses that when the pulse duration is constant, the apparent mobility decreases as the interval between pulses becomes longer. The changes in apparent mobility due to pulse duration and pulse interval are reported in this article to be relatively small for short fragments of 3,400 base pairs, and quite large for longer fragments of 10,000 base pairs and more. In addition, it is indicated in this article that the dependence of the mobility on pulse interval and duration decreases with decreasing ion concentration in the gel (the authors varied the sodium ion concentration between 0.04 to 0.4 M); and these effects become larger with decreasing pore size in agarose. Further, the article presents some mathematical analysis concerning the reasons for the observed greater effects on larger molecules, but provides no quantitative information related to DNA fragments containing more than 22,000 base pairs. Also, no mention is presented in this article relating to the mathematical analysis as a guide to a process for separating large DNA fragments by choosing optimal experimental conditions for a given mixture of fragments.
In "Prediction of Chain Elongation in the Reptation Theory of DNA Gel Electrophoresis," Biopolymers, Vol. 24, No. 12, pages 2181-2184 (1985), the disclosure of which is totally incorporated herein by reference, G. W. Slater and J. Noolandi provide a theoretical discussion of the reptation theory of DNA chain motion with respect to gel electrophoresis. This article discloses three time scales which are used in calculating optimal experimental conditions for the method of the present invention; it does not, however, provide a full quantitative analysis of the correlation between the time scales, the duration of applied field pulses, and the sizes of DNA fragments to be separated. A detailed quantitative analysis is provided in "On the Reptation Theory of Gel Electrophoresis," G. W. Slater and J. Noolandi, Biopolymers, Vol. 25, No. 3, pages 431-454 (Mar. 1986), the disclosure of which is totally incorporated herein by reference, and this analysis is important for the purpose of understanding and/or deriving a basis of the present invention.
Many references disclose the basic process of gel electrophoresis. For example, U.S. Pat. No. 3,630,882 teaches an apparatus for particle separation wherein a mixture of particles in a suspending medium is subjected to an intermittent DC electrical field of sufficient strength to produce a sharp separation of two or more components of the mixture. The electric field is intermittent or pulsed so that the particles in the material are alternately subjected to high electric field and low or zero electric field.
Also, U.S. Pat. No. 3,870,612 teaches a method of determining the electrophoretic mobility and diffusion coefficient of a macromolecular polymer in solution wherein the macromolecules are driven through the solution by an electric field in a modified electrophoretic cell. The electric field is pulsed, and the pulses are of alternating polarity to allow for the use of high fields and to prevent formation of concentration gradients.
Further, in U.S. Pat. No. 3,930,982 there is disclosed an apparatus for generating a periodic non-uniform electric field for the purpose of removing polarizable particulate material from a liquid by dielectrophoresis. The liquid containing particles to be removed is passed over a ferroelectric apparatus, which generates a periodic non-uniform electric field near the boundary between alternately polarized portions of the ferroelectric material, which periodic non-uniform electric field is generated by subjecting portions of the ferroelectric material to an alternating potential to alternately polarize the portions, while allowing other portions of the ferroelectric material to remain polarized in the same direction.
In addition, in U.S. Pat. No. 4,148,703 there is disclosed a method of electrophoretic purification of electrically charged biomolecules which uses different geometrically shaped electrode configurations, permitting potentially different gradients and enabling different particle velocities, finer separations, and continuous electrophoresis by means of a higher voltage in a smaller area, with a decrease in power expenditure. The various electrode systems are alternately turned on and off at a given time independently of one another and for a given duration of time; and in U.S. Pat. No. 3,506,554, there is illustrated a process and apparatus for separating electrophoretically active substances, such as proteins. The method utilizes a continuously flowing stream of buffer to transport the substances through a zone having an inert material that is pereable to either the electrophoretically active material or small buffer ions, such as a polyacrylamide gel slab. The process includes applying an electric field first in one direction and then in another direction to enable separation, and the cycle of reversing the direction of the electric field is repeated many times.
There is disclosed in U.S. Pat. No. 4,061,561 an electrophoresis apparatus that allows for high resolution by performing two dimensional migrations in a square tray. The sample selected is subjected to a linear current in one direction, and the tray is then turned exactly ninety degrees so that the first migration is pulled apart from an orthogonal direction. Also, the '561 patent discloses a multiple-sample applicator that allows an operator to deposit multiple samples on the gel or membrane either simultaneously or one at a time.
A process and apparatus for purifying and concentrating DNA from a crude DNA - containing mixture, such as whole blood, is disclosed in U.S. Pat. No. 4,617,102. The apparatus of the '102 patent consists essentially of an agarose gel disc immersed in an electrophoresis buffer solution and supported between two eight-micrometer polycarbonate filters in an electric field. Placing the sample on the disc and applying an electric field results in the separation of the DNA from the other components of the crude mixture. However, the reference does not, for example, teach a method of separating DNA particles of different molecular weights from each other.
Other references of interest include U.S. Pat. No. 4,322,275; "Fractionation of Large Mammalian DNA Restriction Fragments Using Vertical Pulsed - Field Gradient Gel Electrophoresis," K. Gardiner, W. Laas, and D. Patterson, Somatic Cell and Molecular Genetics, Vol. 12, No. 2, pages 185-195 (1986); "Mapping of the Class II Region of the Human Major Histocompatibility Complex by Pulsed - Field Gel Electrophoresis," D. A. Hardy et al., Nature, Vol. 323, pages 453-455 (1986); and "New Biased - Reptation Model for Charged Polymers," G. W. Slater and J. Noolandi, Physical Review Letters, Vol. 55. No. 15, pages 1579-1582 (1985).
Current methods used for separation of DNA fragments having more than 20,000 base pairs have several disadvantages. In some instances, commercially available electrophoresis equipment must be modified before these methods can be applied. For example, the process disclosed in the '452 patent involving the use of crossed gradient fields, requires extensive alterations to conventional gel electrophoresis apparatus. Also, all of the above described systems intended for separating DNA fragments of more than 20,000 base pairs use relatively high electric fields (above 3 volts/cm) necessitating the implementation of a bulky and expensive cooling system to avoid degradation of the gel and/or the DNA, whereas in one embodiment of the present invention the electric field may be as low as 0.25 volts/cm. In addition, no method currently known provides a reliable way of determining in advance the values of experimental parameters that must be selected to obtain optimal resolution of a given mixture of DNA fragments. Optimal resolution may be defined as obtaining results wherein most or all of the fragments of a particular size may be found in a distinct band that does not overlap with bands of fragments of another size on the termination of the process. Further, the existing methods of separation often lead to non-reproducible results, a disadvantage alleviated with the process of the present invention.